It is known that when oligo- and polysaccharides are subjected to esterification by conventional methods, products are formed which exhibit a random distribution of the esters groups within the AGU and along the chain. This randomness depends on the accessibility to electrons or on the spatial accessibility of the individual hydroxyl groups. Thus in starch, for example, the hydroxyl group in the C6 position is a primary group which is very sterically accessible and which thus exhibits the highest accessibility during heterogeneous reactions in particular. The hydroxyl function of the C2 position forms the basis of the electronic effect of the adjacent glycoside bond and of the electron-attracting properties of the ring oxygen. In homogeneous processes, it has been shown that the C2 position reacts first for this reason. In none of the aforementioned situations, however, is the complete reaction of only one hydroxyl group achieved.
By utilising differences in accessibilities such as these, hydroxyl groups can be selectively blocked with the aid of protective groups, which are generally bulky and can easily be split off, so that regioselective derivatives are formed in subsequent reactions.
Examples of protective groups such as these include triphenylmethyl groups or bulky organosilicon entities such as t-hexyl- or tert-butyl-dimethylsilyl groups.
However, this type of synthesis has the decisive disadvantage that at least two additional reaction steps are necessary due to the introduction of the protective group and the separation thereof. Other disadvantages are the fact that the separation of the protective groups is sometimes incomplete, which means that the cleavage products which are formed thereby, and which are sometimes toxic, have to be removed without leaving a residue, as well as the breakdown of the polysaccharide chain which is possible under the conditions of cleavage and which changes the properties of the product.
When a plurality of reactive centres, e.g. hydroxyl groups, exists in a molecule, enzymes are capable of catalysing direct, selective esterification reactions. In this connection, each enzyme has a certain folded (native) structure which is essential for its specific biocatalytic activity in the physiological medium concerned. It has been shown in numerous publications, however, that many enzymes are also active in organic solvents, i.e. are active irrespective of their native structure, size and function. Enzymes generally exhibit a high activity in nonpolar solvents, whereas only very low activities are found in relatively polar media (Biotechnol. Bioeng. 30 (1987), 81-87). Enzymes are insoluble in the latter organic solvents.
Numerous enzyme-catalysed reactions of this type have already been carried out on low molecular weight mono- and disaccharides in organic solvents (FEMS Microbiol. Rev. 16 (1995), 193-211; J. Prakt. Chem. 335 (1993), 109-127; Synthesis 1992, 895-910; WO 97/36000; WO 95/23871). In these reactions lipases have primarily been used as the enzymes, although esterases and proteases have also been used, and solvents such as tetrahydrofuran, pyridine and N,N-dimethylformamide have been employed. Enzymatic esterification can also be effected in an aqueous buffer solution. One disadvantage here is that the acylating reagent, which has a character similar to that of a fatty acid, is insoluble in the aqueous buffer solution and can thus only be suspended therein. The substrate which is to be esterified is present in dissolved form (DE-A-34 30 944, 1992; JP-A-63191802).
Glucans generally result in the formation of esters at primary hydroxyl groups. In some cases, esterification also occurs at secondary hydroxyl groups (J. am. Chem. Soc. 109 (1987), 3977-3981; Enzyme Microb. Technol. 20 (1997), 225-228). Compounds which comprise electron-attracting groups are generally used for the aforementioned enzymatic esterifications, such as vinyl esters (Biotechnol. Lett. 19 (1997), 511-514) or trihalogenoethyl esters (Tetrahedron 54 (1998), 3971-3982); esterification by a vinyl ester constitutes an irreversible reaction, since the vinyl alcohol which is formed is removed as acetaldehyde from the reaction equilibrium. Other reactive compounds include carboxylic anhydrides and esters of carbonic acid.
Diesters of dicarboxylic acids have also been used for reaction with mono- and disaccharides. In this manner, it has proved possible to synthesise new saccharide-based copolymers, as disclosed in U.S. Pat. No. 5,270,421 and U.S. Pat. No. 5,618,933.
In the publications described above, the substrate and the acylating reagent are generally dissolved in an organic solvent, in which the enzyme is then suspended. Enzymatic esterifications cannot be effected on polysaccharides, particularly glucans, by the methods cited above, since the undissolved or unswollen polymer is not accessible to the enzyme or to enzyme catalysis. Polysaccharides can be esterified enzymatically in a heterogeneous phase, however (WO 96/13632, DE-A-34 30 944). These heterogeneous reactions only proceed at the surface of the polymer particles, due to which inhomogeneously esterifed polysaccharide derivatives are formed, i.e. polysaccharide derivatives which comprise a non-uniform distribution of substituents along the polymer chain. Other disadvantages of this method of esterification are that low yields of product are obtained and products are formed which comprise a low degree of selectivity as regards the type of substitution in the anhydroglucose unit.
Conventional heterogeneous esterifications are often conducted in water as a suspension medium (U.S. Pat. No. 5,703,226, 1997). Homogeneous reactions are conducted in organic solvents or in the acylating reagent directly (U.S. Pat. No. 5,714,601, 1998; WO 96/14342). The corresponding carboxylic anhydrides or vinyl esters are generally used as acylating agents. Esterifications or transesterifications of this type are mostly catalysed by alkalies, wherein suitable catalysts include alkali hydroxides, salts of mineral acids or organic amines.